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Molecular Endocrinology, doi:10.1210/me.2008-0055
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Molecular Endocrinology 22 (10): 2307-2321
Copyright © 2008 by The Endocrine Society

Prolactin and ErbB4/HER4 Signaling Interact via Janus Kinase 2 to Induce Mammary Epithelial Cell Gene Expression Differentiation

Rebecca S. Muraoka-Cook, Melissa Sandahl, Debra Hunter, Leah Miraglia and H. Shelton Earp, III

University of North Carolina Lineberger Comprehensive Cancer Center (R.S.M.-C., M.S., D.H., L.M., H.S.E.), Departments of Genetics (R.S.M.-C.), Medicine (H.S.E.), and Pharmacology (H.S.E.), University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: H. Shelton Earp III, University of North Carolina Lineberger Comprehensive Cancer Center, University of North Carolina Chapel Hill, 102 Mason Farm Road, Chapel Hill, North Carolina 27599. E-mail: hse{at}med.unc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Differentiation of mammary epithelium in vivo requires signaling through prolactin and ErbB4/HER4-dependent mechanisms. Although stimulation of either the prolactin receptor or ErbB4/HER4 results in activation of the transcription factor signal transducer and activator of transcription 5A (STAT5A) and induction of lactogenic differentiation, how these pathways intersect is unknown. We show herein that prolactin signaling in breast cells cooperates with and is substantially enhanced by the receptor tyrosine kinase ErbB4/HER4. Prolactin and the ErbB4/HER4 ligand heparin-binding epidermal growth factor each induced STAT5A tyrosine phosphorylation and nuclear translocation; each pathway required the intracellular tyrosine kinase Janus kinase 2 (JAK2). We found that full prolactin-mediated STAT5A activation and binding to the endogenous β-casein promoter required ErbB4/HER4 but did not require ErbB1/epidermal growth factor receptor. For example, prolactin- induced STAT5A activity was markedly diminished in cells overexpressing kinase inactive HER4, in cells transfected with small interfering RNAs to specifically knock down endogenous ErbB4/HER4 expression and in cells treated with a small molecule inhibitor that targets ErbB4 kinase. Interestingly, prolactin caused ErbB4/HER4 tyrosine phosphorylation in a JAK2 kinase-dependent manner. Finally, prolactin receptor, ErbB4/HER4, and JAK2 were coimmunoprecipitated from prolactin-treated but not untreated cells. These results suggest that prolactin signaling engages the ErbB4 pathway via JAK2 and that ErbB4 provides an important component of STAT5A-dependent lactogenic differentiation; this pathway integration may help explain the similar deficit in mammary development observed in gene-targeted mice deficient in prolactin receptor, JAK2, ErbB4, or STAT5A


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GROWTH FACTOR and hormone signaling networks regulate the physiological cycles of proliferation, differentiation, and involution in the mammary epithelium. Dysregulation of these signaling networks via multiple mechanisms may produce pathological consequences including cancer (1). Prolactin (PRL) is a polypeptide hormone primarily synthesized in the anterior pituitary and acts systemically as a classical endocrine factor. Additional evidence suggests that PRL is produced locally by the mammary epithelium, placenta, uterus, bone, brain, and immune system and can act in an autocrine and/or paracrine fashion (2, 3). In breast epithelial and cancer cells, PRL stimulates growth, development, and differentiation by signaling through the PRL receptor (PRLR), a member of the cytokine receptor family (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Upon PRL binding, the PRLR couples to the nonreceptor tyrosine kinase, Janus kinase 2 (JAK2), which then phosphorylates target proteins, activating downstream signaling pathways (5, 9, 14, 15, 16, 17). PRL-mediated JAK2 activity engages the ERK (p44/42ERK) and signal transducer and activator of transcription (STAT) signaling pathways, with STAT5A being the principal STAT isoform whose tyrosine phosphorylation, dimerization, and nuclear translocation help mediate PRL-dependent differentiation of mammary epithelial cells during pregnancy and lactation (5, 12, 16, 18, 19, 20). PRL and GH, a related pituitary cytokine with similar signaling mechanisms, have each been reported to cooperatively signal with the epidermal growth factor (EGF) receptor (EGFR) (13, 21, 22, 23, 24).

EGFR is a member of the ErbB family of receptor tyrosine kinases. Each ErbB family member regulates different aspects of mammary development (25, 26). The four family members, EGFR/ErbB1/HER1, ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4, each contain an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. The ErbB receptors exhibit ligand-inducible dimerization, transphosphorylation, and tyrosine kinase activation, with the exception of the catalytically inactive ErbB3. Homo- or heterodimer formation between ErbB family members occurs upon ligand binding. ErbB2 is the preferred heterodimeric partner although ErbB2 does not bind any conventional ligand, relying on heterodimerization with liganded ErbB1, ErbB3, or ErbB4 for tyrosine kinase induction. In the breast epithelium, the ligands EGF, amphiregulin, and TGF-{alpha} bind exclusively to EGFR, producing signals that enhance proliferation but not differentiation (25, 27). Heparin-binding-EGF (HB-EGF) binds to both EGFR and ErbB4 and contributes to differentiation primarily through ErbB4. Similarly, heregulin (HRG, also known as neuregulin-1) binds to ErbB3 and ErbB4 and contributes to differentiation primarily via ErbB4 (28, 29, 30, 31, 32).

Multiple mouse models have demonstrated that ErbB4 signaling is required for lactogenesis and differentiation of murine mammary glands (33, 34, 35). In vivo, ErbB4 expression and activity are lowest during phases of epithelial cell proliferation (puberty and early pregnancy) and highest during phases of differentiation (late pregnancy and early lactation) (36). Mammary glands from mice that lack ErbB4 activity have lactational defects due to an impaired program of differentiation, measured by decreased expression of milk proteins and decreased activity of STAT5A, a transcription factor required for the expression of genes encoding milk proteins (33, 34, 35). STAT5A deficiency also results in failed lactogenesis, a phenotype reminiscent of ErbB4-deficient mammary glands (18, 19, 20).

ErbB4 is the only receptor tyrosine kinase cleaved by a two-step proteolytic process releasing an 80-kDa intracellular domain with an active tyrosine kinase and nuclear localization capabilities (37, 38, 39, 40). Studies performed in cell culture have shown that the soluble 80-kDa intracellular domain of ErbB4 can directly associate with STAT5A in breast epithelial cells and can be detected in complex with DNA-bound STAT5A (41, 42, 43). This suggests that the intracellular domain of ErbB4 may act as a transcriptional coactivator for STAT5A.

Although accumulating evidence suggests that ErbB4 is required for differentiation of mammary epithelial cells, the molecular mechanism by which ErbB4 directs differentiation is not well understood, particularly in regard to the overlapping signaling cascade constituents shared with the pathway initiated by PRL. In this report, we describe experiments using the mouse mammary epithelial cell line HC11 as a model of mammary differentiation to investigate ErbB4 and its role in PRL-mediated differentiation. These data demonstrate that, in addition to converging on STAT5A to induce differentiation, PRL results in ErbB4 tyrosine phosphorylation in a JAK2-dependent manner and that ErbB4 kinase activity maximizes differentiation in response to PRL.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ErbB4 Ligand HB-EGF Increases PRL-Induced STAT5A Activity
We used the HC11 cell line, derived from the mammary gland of a female BALB/C mouse at midpregnancy, as a model of mammary epithelial cell differentiation. We have previously shown that ErbB4 activity results in STAT5A phosphorylation, nuclear translocation, and transcriptional activity in HC11 cells (32). Because STAT5A activation is a required aspect of lactogenic differentiation of the mammary epithelium, it is often used as a molecular marker of differentiation. Because activation of the PRLR and ErbB4 both result in STAT5A activation and both are required for differentiation of the mammary epithelium in vivo, we examined the combined effects of PRLR activation and ErbB4 activation on STAT5A-dependent transcription. Ligand-induced transcriptional activation was examined in transient transfection assays using a reporter construct comprised of residues (–294 to +1) of the human β-casein gene driving the Photinus luciferase cDNA (pβcasein-lux). This region of the β- casein gene harbors STAT5A DNA-binding sites (44) and has been used previously as a STAT5A-dependent reporter (32, 45). Treatment of HC11 cells for 48 h with PRL (500 ng/ml) increased luciferase activity 6.2-fold over untreated HC11 cells, whereas HB-EGF treatment (2 nM) resulted in a 5.1-fold increase (Fig. 1AGo). Although HB-EGF binds to both EGFR and ErbB4 in HC11 cells, we and others have previously shown that differentiation of HC11 cells does not occur in the context of activated EGFR activity (27, 32). Combined treatment with PRL and HB-EGF for 48 h increased transcription from this STAT5-dependent reporter 16.4-fold over untreated cells, a level more than twice what is seen with either HB-EGF (P < 0.01, Student’s t test) or PRL (P < 0.01, Student’s t test) alone and greater than the sum of PRL- and HB-EGF-induced levels. Thus, the two signals are at least additive.


Figure 1
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  		Fig. 1. ErbB4 and PRL Synergize to Activate STAT5A

A, HC11 cells transiently transfected with pβcasein-lux were treated 48 h with PRL (500 ng/ml) or HB-EGF (2 nM) or both together. Cells were harvested, and lysates were assayed for luciferase activity and protein concentration. Values are presented as relative light units (RLU) per microgram total protein. Values represent the average of three independent experiments, each one analyzed in triplicate. Error bars represent SD. B, HC11 cells were stably transfected with pcDNA4 vector or pcDNA4-HER4, encoding human ErbB4/HER4. Pooled clones of exponentially growing cells were used for immunoblot (IB) of ErbB4, ErbB1, and PRLR; for immunoprecipitation (IP) of JAK2 followed by IB for either JAK2 or phosphotyrosine 1007/1008 JAK2 (P-JAK2); or for immunoprecipitation of STAT5A followed by IB for either STAT5A or phosphotyrosine STAT5A/B (P-STAT5). C, STAT5A was immunoprecipitated from cross-linked HC11-HER4 and HC11-HER4KD cells cultured 48 h in serum-free medium and then treated with or without PRL for 30 min. PCR to detect mouse β-casein promoter sequences was performed on whole-cell lysates and on STAT5A immunoprecipitates. D, Pooled clones of HC11-pcDNA4, HC11-HER4, or HC11-HER4KD cells were transfected with pβcasein-lux and then treated with PRL, HB-EGF, or both for 24 h, measuring luciferase activity as described above. E and F, HC11 cells were transfected with pβcasein-lux and then treated with or without 0.1 nM HB-EGF in the presence of increasing concentrations of PRL (E) or were treated with or without 10 ng/ml PRL in the presence of increasing concentrations of HB-EGF (F). Luciferase activity was measured as described above.

 
Overexpression of ErbB4/HER4 Increases PRL Signaling in HC11 Cells
We tested the hypothesis that PRL synergizes with ErbB4 to induce differentiation, even in the absence of ErbB4 ligand activation. We expressed a human HER4 (Jma-Cyt1 isoform) cDNA construct in HC11 cells by stable transfection (HC11-HER4 cells). Pooled clones of selected cells were serum starved and then treated for 1 h with PRL. Whole-cell extracts were examined for site-specific STAT5A and JAK2 tyrosine phosphorylation. Total STAT5A and JAK2 protein levels were equal across each HC11 cell line tested, regardless of ligand addition (Fig. 1BGo). Although PRL treatment induced phosphorylation of STAT5A on Tyr 694 and phosphorylation of JAK2 on Tyr 1007/1008 in HC11, HC11-pcDNA4, and HC11-HER4 cells, we found a greater level of PRL-induced STAT5A and JAK2 tyrosine phosphorylation in cells overexpressing ErbB4/HER4 as compared with cells expressing the empty pcDNA4 vector or parental HC11 cells. This suggests that ligand activation of ErbB4 is not required for ErbB4 to enhance PRL-mediated signaling to downstream targets such as STAT5A and JAK2.

Cooperation between PRL and ErbB4 Signaling Requires ErbB4/HER4 Kinase Activity
PRL-induced binding of STAT5A to the endogenous β-casein promoter was examined in HC11-HER4 cells by chromatin immunoprecipitation (ChIP) PCR analysis. Using primer sequences flanking the STAT5A binding site within the 5' flanking region of the mouse β-casein gene, PCR analysis demonstrated that STAT5A coprecipitated with the mouse β-casein promoter in cross-linked HC11-HER4 cells treated with PRL (Fig. 1CGo). However, STAT5A was not bound to the β-casein promoter region in PRL-treated HC11-HER4KD cells, which overexpress kinase-dead human HER4.

STAT5A-induced transcriptional activity was examined in HC11-HER4 cells treated with PRL, HB-EGF, or both. Treatment of HC11-HER4 cells with PRL alone increased STAT5A-dependent transcription by 9.5-fold over untreated HC11-HER4 cells, compared with a 5.8-fold increase in response to PRL in HC11-pcDNA4 cells over untreated HC11-pcDNA4 cells (Fig. 1DGo). In HC11-HER4KD cells, HB-EGF-induced transcriptional activation was abolished and PRL-induced transcriptional activation was clearly diminished, in agreement with the reduced STAT5A binding to the endogenous β-casein promoter (Fig. 1CGo). Similar to parental HC11 cells, treatment of HC11-pcDNA4 cells with PRL and HB-EGF together resulted in a greater than additive activation of this STAT5-dependent promoter (15.2-fold using HB-EGF and PRL together, vs. 5.8- or 4.9-fold using PRL or HB-EGF alone, respectively). However, HC11-HER4 cells treated with both PRL and HB-EGF displayed a 23.8-fold increase in luciferase reporter activity.

HC11 cells transfected with pβcasein-lux were treated with suboptimal levels of HB-EGF (0.1 nM) along with increasing concentrations of PRL (Fig. 1EGo); in addition, cells were treated with suboptimal levels of PRL (10 ng/ml) and increasing concentrations of HB-EGF (Fig. 1FGo). STAT5A-dependent reporter activity was examined, revealing that the level of reporter activity in response to combined PRL and HB-EGF was always greater than the sum of the reporter activities of each ligand (Fig. 1EGo). These studies suggest that ErbB4/HER4 signaling enhances the activation of STAT5A-dependent transcription in response to PRL.

ErbB4 Is Required for JAK2 Activation in Response to HB-EGF But Not PRL
Treatment of serum-starved HC11 cells with HB-EGF induced tyrosine phosphorylation of both ErbB4 and JAK2 within 10 min; the effect was sustained through 24 h (Fig. 2AGo). STAT5A activation was observed within 10 min of HB-EGF treatment. Because HB-EGF activates both ErbB1 and ErbB4, we suppressed the expression of ErbB4 in HC11 cells using small interfering RNA (siRNA) SMARTpool sequences directed against mouse ErbB4 to determine whether ErbB4 is required for JAK2 activation. Transient transfection of cells with ErbB4 siRNA sequences substantially reduced ErbB4 expression and ligand-induced ErbB4 activation as detected by Western analysis (Fig. 2BGo), whereas cells transfected with a scrambled siRNA sequence displayed tyrosine phosphorylation of ErbB4 in response to both HB-EGF and PRL. Expression of ErbB1 was unaffected by transfection with scrambled or anti-ErbB4 siRNA sequences (data not shown). JAK2 activation (Tyr 1007/1008 phosphorylation) was detected in response to HB-EGF and to PRL in cells transfected with the control siRNA sequences. However, tyrosine phosphorylation of JAK2 was not observed in response to HB-EGF in cells treated with ErbB4 siRNA, even though PRL-mediated induction of JAK2 activity was observed in cells lacking ErbB4 expression due to ErbB4 siRNA. These results confirm that ErbB4 activates JAK2 in response to HB-EGF. Although PRL fully induced JAK2 tyrosine phosphorylation, the subsequent STAT5A phosphorylation was diminished in the absence of ErbB4.


Figure 2
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  		Fig. 2. ErbB4 Is Required for JAK2 Activation in Response to HB-EGF But Not PRL

A, HC11 cells were serum starved overnight and then treated for 10 min to 24 h with HB-EGF. Extracts were immunoprecipitated (IP) with antibodies against JAK2 or ErbB4. Immunocomplexes were analyzed by immunoblot (IB) with indicated antibodies. B, HC11 cells were transiently transfected with siRNA sequences directed against mouse ErbB4 (10 µM) or scrambled siRNA sequences (10 µM). Transfected cells were maintained in serum-free medium for 30 h before treatment with either PRL or HB-EGF for 4 h. Cell extracts were analyzed by immunoprecipitation (IP) followed by immunoblot (IB) using the antibodies indicated at the right. C, HC11 cells were transiently transfected with siRNA sequences directed against mouse ErbB4 (10 µM) or scrambled siRNA sequences (10 µM). Transfected cells were maintained in serum-free medium in the presence of either PRL or HB-EGF for 30 h. Cell extracts were analyzed by for luciferase activity. Values are presented as relative light units (RLU) per microgram total protein and represent the average of three independent experiments, each analyzed in triplicate. Error bars represent the SD. D, STAT5A was immunoprecipitated from cross-linked HC11 cells transfected with siRNA sequences targeting ErbB4 or scrambled siRNA sequences (control). Cells were cultured 48 h in serum-free medium and then treated with or without PRL or HB-EGF for 30 min. PCR to detect mouse β-casein promoter sequences was performed on whole-cell lysates and on STAT5A immunoprecipitates.

 
ErbB4 Expression Is Required for PRL-Induced STAT5A Activity
Using cotransfection of the pβcasein-lux reporter construct in cells transfected with scrambled or ErbB4-targeted siRNA sequences, we were able to measure STAT5A-mediated transcription in the absence of ErbB4 expression. Cells transfected with the scrambled siRNA displayed a 4.8-fold and 3.9-fold increase in luciferase activity upon treatment with PRL and HB-EGF, respectively, as compared with untreated, scramble siRNA-transfected cells (Fig. 2CGo). Not surprisingly, HB-EGF treatment was unable to increase luciferase activity over basal levels in cells transfected with ErbB4-directed siRNA. However, PRL treatment also did not increase luciferase expression from this reporter in the absence of ErbB4 expression.

Consistent with these results, ChIP analysis of HC11 cells transiently transfected with scrambled siRNA sequences revealed that STAT5A was bound to the endogenous β-casein promoter in response to either HB-EGF or PRL (Fig. 2DGo). However, STAT5A did not coprecipitate with β-casein promoter sequences in HB-EGF or PRL-treated HC11 cells transfected with ErbB4 siRNA, confirming that ErbB4 expression is required for the series of steps needed to achieve STAT5A binding to specific promoter DNA sequences. Taken together, these data suggest that ErbB4 is not necessary for PRL-mediated induction of JAK2 activity but is required for PRL-mediated induction of full STAT5A activity. These data are consistent with previous reports suggesting that ErbB4 is required for STAT5A activity (32, 33, 34, 35, 41) and suggest that PRL uses a signaling cascade involving both JAK2 and ErbB4 as downstream signaling components to activate STAT5A.

ErbB4 and PRLR Both Require JAK2 to Activate STAT5A
Although JAK2 is required for tyrosine phosphorylation and activation of STAT5A in response to PRL (46, 47, 48), recent reports suggest that STAT5A interacts directly with ErbB4 (41, 43). Because ErbB4 harbors tyrosine kinase activity, it is possible that STAT5A Tyr 694 phosphorylation, and therefore activation of STAT5A, could occur via ErbB4 independently of JAK2. Therefore, we examined whether JAK2 is involved in ErbB4-mediated STAT5A activation. To determine whether JAK2 activity is required for ErbB4 signaling to STAT5A, we used transient transfection assays, in which wild-type JAK2 or kinase-dead JAK2KD were overexpressed in HC11 cells. Transient transfection of either wild-type or kinase-dead JAK2KD resulted in similar levels of total JAK2 expression. However, PRL-mediated JAK2 tyrosine phosphorylation was abolished in HC11 cells expressing kinase-dead JAK2KD (Fig. 3AGo). Cells were cotransfected with pβcasein-lux to assess ligand-induced STAT5A transcriptional activity in the context of wild-type or kinase-dead JAK2 (Fig. 3BGo). Although expression of wild-type JAK2 allowed for inducible STAT5A-mediated transcription in response to PRL or HB-EGF, transient expression of kinase-dead JAK2KD eliminated inducible STAT5A-mediated transcription from pβcasein-lux in response to PRL, as previously described (46, 47, 48), but also in response to HB-EGF. Because HB-EGF activates ErbB4 signaling to induce STAT5A (32), these results demonstrate that JAK2 is required for ErbB4-induced STAT5A activation.


Figure 3
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  		Fig. 3. Kinase Activity and Expression of JAK2 Is Required for ErbB4-Mediated STAT5A Activation

A, HC11 cells were transiently transfected with pRK5-JAK2 or pRK5-JAK2kd. Whole-cell extracts were harvested 48 h after transfection and analyzed by immunoprecipitation (IP) of JAK2 followed by immunoblot (IB) using antibodies to phospho-JAK2 or total JAK2. B, HC11 cells were transfected with pβcasein-lux and cotransfected with pRK5-JAK2 or pRK5-JAK2KD. Cells were cultured 48 h in serum-free medium with or without PRL or HB-EGF. Luciferase activity is shown as the average relative light units (RLU) per microgram total protein ± SD. Experiments were repeated three times, with each sample being analyzed in triplicate. C, HC11, HC11-JAK2{alpha}s, and HC11-JAK2vs cells were serum starved for 16 h and then treated with or without HB-EGF for 4 h. Whole-cell extracts were analyzed by immunoprecipitation (IP) followed by immunoblot (IB) using the antibodies indicated at right. D, HC11, HC11-JAK2{alpha}s, and HC11-JAK2vs cells were transiently transfected with pβcasein-lux and cultured 48 h with PRL (500 ng/ml) or HB-EGF (2 nM). Cells were harvested, and lysates were assayed for luciferase activity and protein concentration. Values are presented as relative light units (RLU) per microgram total protein. Values represent the average of three independent experiments, each one analyzed in triplicate. Error bars represent SD. E, STAT5A was immunoprecipitated from cross-linked HC11, HC11-JAK2vs, and HC11-JAK2{alpha}s cells cultured 48 h in serum-free medium and then treated with or without HB-EGF for 30 min. PCR to detect mouse β-casein promoter sequences was performed on whole-cell lysates and on STAT5A immunoprecipitates.

 
To confirm these findings, we used a stably transfected HC11 line lacking expression of JAK2, accomplished by expression of antisense JAK2 (HC11-JAK2{alpha}s cells), as previously described (17). As a control, a stable cell line expressing a scrambled JAK2 antisense sequence was used (HC11-JAK2vs cells). Western analysis demonstrated reduced expression of JAK2 in HC11-JAK2{alpha}s cells as compared with HC11-JAK2vs cells or to parental controls (Fig. 3CGo). We treated each serum-starved cell line with HB-EGF, assessing STAT5A activity by Western analysis. We found phospho-STAT5A in HB-EGF-treated HC11 cells and HC11-JAK2vs cells but not in HC11-JAK2{alpha}s cells. STAT5A-dependent transcriptional activity was examined in HC11-JAK2{alpha}s and HC11-JAK2vs cells treated with HB-EGF (Fig. 3DGo). Although HB-EGF treatment resulted in 5.0-fold and 6.8-fold increased levels of luciferase activity in parental HC11 and HC11-JAK2vs cells, respectively, we found that loss of JAK2 expression in HC11-JAK2{alpha}s cells impaired STAT5A-dependent transcription in response to HB-EGF. ChIP analysis of HB-EGF-treated HC11 and HC11-JAK2vs cells showed that STAT5A was bound to the mouse β-casein promoter in response to HB-EGF (Fig. 3EGo). However, STAT5A did not coprecipitate with the mouse β-casein promoter in HC11-JAK2{alpha}s cells. These data confirm that JAK2 expression and kinase activity are required for ErbB4-mediated induction of STAT5A.

PRL Induces Tyrosine Phosphorylation of ErbB4/HER4
To address the mechanism by which PRL signaling synergizes with ErbB4 signaling, we investigated the effects of PRL on ErbB4 tyrosine phosphorylation. Treatment of serum-starved HC11 cells with PRL resulted in ErbB4 tyrosine phosphorylation as early as 10 min after stimulation (Fig. 4AGo). PRL-induced tyrosine phosphorylation of ErbB1 was also noted, as previously reported by others (21, 23). Although PRL-induced ErbB1 tyrosine phosphorylation rapidly diminished by 30 min, PRL-induced ErbB4 activation was sustained for at least 4 h. Pretreatment of HC11 cells for 16 h with the small molecule inhibitor GW572016 (lapatinib; 10 µM) impaired tyrosine phosphorylation of ErbB4 in response to PRL and HB-EGF (Fig. 4BGo). GW572016 did not affect expression of ErbB4. This indicates that some level of initial ErbB4 tyrosine phosphorylation is necessary for subsequent PRL-dependent tyrosine phosphorylation of ErbB4, suggesting that a Src homology 2 (SH2) domain-containing protein may mediate that tyrosine phosphorylation of ErbB4 in response to PRL.


Figure 4
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  		Fig. 4. Tyrosine Phosphorylation of ErbB4 in Response to PRL

A, Western analysis to detect tyrosine phosphorylation of ErbB1 and ErbB4 immunoprecipitates (IP) from serum-starved HC11 cells treated with HB-EGF or with PRL for 10 min to 4 h. B, HC11 cells were serum starved 16 h in the presence or absence of 10 µM GW572016 before treatment with PRL or HB-EGF for 4 h. Extracts were used for immunoprecipitation (IP) and immunoblot (IB) with the antibodies indicated at right. C, 293 cells were transiently transfected with expression constructs encoding HER4/ErbB4, JAK2, or JAK2KD. Cells were cultured in serum-free medium for 48 h. Cell lysates were analyzed directly by IB to detect JAK2 expression or immunoprecipitated with antibodies against ErbB4/HER4 followed by IB to detect either phosphotyrosine or ErbB4. D, HC11 and HC11-JAK2KD cells were treated for 0–96 h with PRL. Whole-cell extracts were analyzed by immunoprecipitation (IP) of ErbB4 followed by immunoblot (IB) with either ErbB4 or phosphotyrosine (P-Tyr). E, HC11 cells were serum starved 16 h with or without GW572016 (10 µM). Cells were then treated with or without PRL for 4 h, and membrane proteins were cross-linked for 20 min with DTSSP. Whole-cell extracts were harvested in 125 mM NaCl and used directly for immunoblot or were used for immunoprecipitation (IP) of ErbB4 or PRLR followed by immunoblot (IB) using the antibodies indicated at the right.

 
JAK2 Is Required for PRL-Induced Phosphorylation of ErbB4
Because JAK2 is an SH2 domain-containing protein that is activated by PRLR and is known to bind to phosphotyrosine residues in the EGFR, we determined whether JAK2 activation results in tyrosine phosphorylation of ErbB4. We transiently coexpressed human HER4/ErbB4 with human JAK2 in 293 cells and found that tyrosine phosphorylation of HER4/ErbB4 occurred in cells coexpressing JAK2 but not in cells without JAK2 or in cells coexpressing kinase-dead JAK2kd (Fig. 4CGo). We examined the requirement for JAK2 activity in PRL-mediated ErbB4 tyrosine phosphorylation, using HC11-JAK2KD cells. Although PRL treatment resulted in tyrosine phosphorylation of ErbB4 in parental HC11 cells as early as 30 min and was sustained through 96 h, PRL-mediated ErbB4 phosphorylation was blocked in cells expressing JAK2kd through 96 h (Fig. 4DGo). Because JAK2 is required for STAT5A induction in response to both PRL and ErbB4 signaling and is required for ErbB4 tyrosine phosphorylation of ErbB4 in response to PRL, these results are consistent with the hypothesis that JAK2 is the point at which PRLR and ErbB4 signaling pathways begin to intersect to enhance STAT5A signaling and differentiation of mammary epithelial cells.

PRLR-JAK2-ErbB4 Complex Formation in PRL-Treated Cells
PRL-treated HC11 cells were used to determine whether ErbB4, JAK2, and PRLR could be detected in a protein complex in cells. ErbB4 immunoprecipitates from untreated cells did not coprecipitate with either PRLR or JAK2 (Fig. 4EGo). However, PRLR and JAK2 were both detected in ErbB4 immunoprecipitates from PRL-treated cells. This complex formation depended on ErbB kinase activity, because pretreatment with the ErbB kinase inhibitor GW572016 for 16 h before the addition of PRL impaired the PRL-dependent ErbB4-PRLR coprecipitation. Converse experiments performed by immunoprecipitation of PRLR gave similar results, demonstrating that the PRLR-ErbB4-JAK2 complex could be coprecipitated from PRL-treated cells but not from untreated cells or PRL-treated cells lacking ErbB4 kinase activity.

ErbB1 Is Not Required for PRL-Mediated ErbB4 Phosphorylation or for PRL-Induced STAT5A Activation
ErbB1/EGFR/HER1 is expressed in HC11 cells and is required for their EGF-induced growth but not for differentiation in response to HB-EGF (32). However, previous reports have demonstrated that ErbB1/EGFR/HER1 signaling may be modified by PRL in breast-derived cells. To determine whether ErbB1/EGFR/HER1 signaling is required for PRL-induced tyrosine phosphorylation of ErbB4/HER4, we used COS7 cells to transiently transfect expression vectors encoding PRLR, JAK2, and STAT5A, along with either HER4 or HER4KD, in the presence or absence of human HER1 or HER1KD. In the absence of HER1, treatment of transfected COS7 cells with PRL for 30 min resulted in tyrosine phosphorylation of HER4 but not of HER4KD (Fig. 5AGo). PRL-induced tyrosine phosphorylation of HER4 occurred in the absence of exogenous HER1 and was unaffected by the presence of exogenous HER1 or HER1KD. Similarly, PRL-induced tyrosine phosphorylation of STAT5A occurred in the presence of HER4, but not in its absence, and not in the presence of HER4KD. Furthermore, HER1 expression in the absence of HER4 expression was insufficient to support PRL-mediated STAT5A activation in COS7 cells. Expression of kinase-dead HER1KD did not affect PRL-mediated STAT5A tyrosine phosphorylation in cells expressing HER4. Therefore, ErbB1/EGFR/HER1 activity may not be required for PRL-induced tyrosine phosphorylation of ErbB4/HER4.


Figure 5
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  		Fig. 5. ErbB1 Is Not Required for PRL-Induced STAT5A Activation

A, COS7 cells were transiently transfected with expression vectors encoding human PRLR, human JAK2, and human STAT5A. Cells were cotransfected with pcDNA3 expression vectors encoding human HER4 or HER4KD in the presence or absence of cotransfected human HER1 or HER1KD. Transfected cells were cultured in medium containing 2% serum. PRL was added for the final 30 min of culture, where indicated. Immunoprecipitation (IP) of whole-cell lysates was performed using antibodies against HER1, HER4, and STAT5. Immunoprecipitates were analyzed by immunoblot (IB) with the indicated antibodies. B, HC11 cells were cultured at confluence for 48 h in serum-free medium. Cells were pretreated with gefitinib (100 nM) for 16 h where indicated and then treated with or without PRL for 30 min. Whole-cell lysates were used for immunoprecipitation (IP) of ErbB1, ErbB4, and STAT5A, followed by immunoblot (IB) with the indicated antibodies. C, STAT5A was immunoprecipitated from cross-linked HC11 cells cultured at confluence for 48 h in serum-free medium and then treated with or without gefitinib (100 nM) or GW572016 (10 µM) for 16 h. Cells were then treated with or without PRL for 30 min. PCR to detect mouse β-casein promoter sequences was performed on whole-cell lysates and on STAT5A immunoprecipitates. D, HC11 cells were cultured at confluence for 48 h in serum-free medium and then treated with or without PRL or HB-EGF for 30 min, and cell membrane proteins were cross-linked with DTSSP. Whole-cell lysates were extracted in low-salt buffer (125 mM NaCl) and then used for immunoprecipitation (IP) with antibodies against ErbB1 or ErbB4. Immunoprecipitates were analyzed by immunoblot for the antibodies indicated at right.

 
To examine the role of ErbB1 in prolactin-mediated differentiation of mouse HC11 cells, we treated HC11 cells with gefitinib, a small molecule kinase inhibitor that is highly specific for ErbB1 and ErbB2 at 100 nM but that does not inhibit ErbB4 activity when used at 100 nM (Fig. 5BGo). Treatment with gefitinib inhibited tyrosine phosphorylation of ErbB1 in response to PRL. However, PRL-induced ErbB4 tyrosine phosphorylation was not affected by gefitinib. Gefitinib treatment did not interfere with PRL-mediated STAT5A phosphorylation, suggesting that kinase activity of ErbB1 is not required for PRL-mediated STAT5A activation. Using ChIP analysis of PRL-treated HC11 cells, it was shown that pretreatment of HC11 cells with gefitinib to inhibit ErbB1 activity did not affect PRL-induced STAT5A DNA binding to the mouse β-casein promoter (Fig. 5CGo). However, STAT5A was not bound to the endogenous β-casein promoter in HC11 cells that were pretreated with the pan-ErbB inhibitor, GW572016 (lapatinib) at 10 µM.

To determine whether ErbB1 localized to the JAK2-ErbB4-PRLR complex, HC11 cells cultured at confluence for 48 h in serum-free medium (supplemented with insulin and hydrocortisone) were treated with PRL or with HB-EGF for 30 min. Cross-linked cells were then used for immunoprecipitation with antibodies against ErbB1 or ErbB4 (Fig. 5DGo). Consistent with previous reports describing a constitutive association between ErbB1 and JAK2, we found ErbB1 and JAK2 bound together under basal serum-free conditions, but ErbB1 did not coprecipitate with PRLR under basal conditions. Under basal conditions, ErbB4 was also found to associate with Jak2 but not with PRLR.

Tyrosine phosphorylation of both ErbB1 and ErbB4 was seen in cells treated with PRL, and both ErbB1 and ErbB4 coprecipitated with PRLR in PRL-treated cells. However, JAK2 was not complexed with ErbB1 in PRL-treated cells but was found in association with ErbB4 in PRL-treated cells. The extent of ErbB4-JAK2 association appeared stronger in PRL-treated cells as compared with untreated cells. This suggests that, although PRL signaling results in the tyrosine phosphorylation of both ErbB1 and ErbB4, JAK2 preferentially associates with ErbB4 in response to PRL.

Activation of ErbB1 and ErbB4 with HB-EGF resulted in tyrosine phosphorylation of both ErbB1 and ErbB4 and the coprecipitation of both PRLR and JAK2 with ErbB1 and with ErbB4. Although the reasons for this are unclear, it is possible that ErbB1-ErbB4 heterodimers induced by HB-EGF allow for the recruitment of JAK2 and PRLR to this ternary complex primarily through the involvement of ErbB4, whereas PRL may induce ErbB4 homodimer formation through JAK2-mediated ErbB4 tyrosine phosphorylation.

PRL-Dependent ErbB4 Tyrosine Phosphorylation Does Not Require c-Src
In addition to JAK2, PRLR activates other signaling pathways that affect phosphorylation of EGFR, including p44/42Erk and c-Src (49, 50). We have confirmed that JAK2, p44/42Erk,and c-Src are each activated in serum-starved HC11 cells upon treatment with PRL (Fig. 6AGo). We investigated the possibility that PRL-induced ErbB4 activation may occur through a mechanism dependent upon c-Src or p44/42Erk. Serum-starved HC11 cells were treated with small molecule inhibitors of c-Src (PP2, 10 µM) or p44/42Erk (U0126, 10 µM) before the addition of PRL, and then ErbB4 tyrosine phosphorylation was examined. PRL-induced phosphorylation of both c-Src and p44/42Erk was inhibited by the addition of the Src inhibitor PP2 or by addition of the Erk inhibitor, respectively. However, PRL-induced phosphorylation of ErbB4 was unaffected by inhibition of c-Src or by inhibition of p44/42Erk. This suggests that c-Src and p44/42 are not required for PRL-induced ErbB4 tyrosine activation.


Figure 6
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  		Fig. 6. PRL-Induced c-Src Activity Is Not Required for Tyrosine Phosphorylation of ErbB4

HC11 cells were cultured at confluence in serum-free medium for 16 h in the presence or absence of PP2 (10 µM), UO126 (10 µM), or GW572016 (10 µM). Cells were then treated with PRL for 4 h, and extracts were harvested. Whole-cell extracts were examined by immunoblot (IB) for total and phospho-p44/42, and total and phospho-Src. Extracts were also used for immunoprecipitation (IP) of ErbB4 followed by immunoblot (IB) for phosphotyrosine (P-Tyr) and total ErbB4. B and C, HC11 cells were cultured at confluence for 48 h in serum-free medium with insulin and hydrocortisone and then transfected with siRNA sequences targeting mouse ErbB4 or scrambled siRNA sequences. Cells were cultured for an additional 48 h in the presence of PRL or HB-EGF, where indicated. Total RNA collected from cells was used to measure relative levels of transcripts encoding SOCS1 (B) and SOCS3 (C) by quantitative real-time RT-PCR. The level of transcripts was calculated using the {delta}{delta}CT method, which normalizes transcript levels of the target RNA to that of a control transcript within the same sample. In this case, transcripts encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference. The level of SOCS1 and SOCS3 transcripts detected in untreated, untransfected cells was set to a value of 1. Values shown represent the average (±SD) of six experiments.

 
We have investigated the effects of PRL and HB-EGF signaling on expression levels of suppressor of cytokine signaling 1 (SOCS1) and SOCS3, proteins known to act as inhibitors of STAT signaling. We have found that 48 h treatment of HC11 cells with PRL did not affect the expression of transcripts encoding either SOCS1 or SOCS3, as measured by quantitative real-time RT-PCR (Fig. 6Go, B and C). However, treatment of HC11 cells with HB-EGF decreased the relative transcript levels of SOCS1 and SOCS3 to approximately 50% of their basal expression levels. Knockdown of ErbB4 expression using siRNA transfection impaired the HB-EGF-mediated decrease in SOCS1 and SOCS3 transcript levels, demonstrating that ErbB4 is required for HB-EGF-induced repression of SOCS1 and SOCS3 gene expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the mammary gland, PRL-mediated STAT5 activity is required for differentiation during pregnancy and lactation, as demonstrated by mouse models targeting the PRLR or STAT5A/B (10, 11, 18, 19, 20). Even heterozygous loss of the PRLR impaired lactation in mice, underscoring the need for PRL signaling. ErbB4 is also required for differentiation and STAT5A activation in the mammary epithelium (33, 34, 35). We report that PRL resulted in sustained ErbB4 tyrosine phosphorylation mediated by JAK2. Because JAK2 is activated by both PRLR and ErbB4, is required for STAT5A activation in response to either PRLR or ErbB4, and is required for PRL-dependent tyrosine phosphorylation of ErbB4, these results suggest that JAK2 is an initial point of convergence for these two signaling pathways. This result is not without precedent because other groups have demonstrated JAK2-dependent phosphorylation of ErbB1 and ErbB2 in response to GH or PRL, respectively (51, 52, 53). These data provide a potential mechanism underlying the observations made in animal models indicating that ErbB4 is required for STAT5A activation during mammary differentiation, even though PRLR signaling to JAK2 remained intact (33, 34, 35).

Additional data using GW572016, the ErbB family inhibitor, suggest that PRL-dependent HER4/ErbB4 phosphorylation also requires the HER4 kinase domain. GW572016 inhibits EGFR and HER2 at submicromolar concentrations, but we and others have shown that 10 µM GW572016 is sufficient to inhibit mouse ErbB4 tyrosine phosphorylation (32). Why the PRL-dependent ErbB4 tyrosine phosphorylation requires both JAK2 and ErbB4 kinase activation is unknown. Perhaps the HER4 kinase is important in bringing JAK2 into proximity to HER4 via SH2-phosphotyrosine interactions. This could be required for the complex formation between JAK2 and ErbB4 (Fig. 5Go), which may be necessary to bring PRLR to ErbB4. Alternatively, GW572016 might alter the conformation of HER4 that both inhibits the HER4 kinase and does not allow a conformation favorable to JAK2 action.

With respect to downstream effects, it is well established that JAK2 is required in mammary epithelial cells for PRL-mediated STAT5 activation (46, 47, 48). We further demonstrate that JAK2 phosphorylation and subsequent STAT5A activation are induced by HB-EGF and that JAK2 is required for HB-EGF-dependent STAT5A transactivation. Despite the direct interaction between STAT5A and the ErbB4 intracellular domain, and despite the tyrosine kinase activity of ErbB4, STAT5A activation by Tyr-694 cannot occur in the absence of JAK2. Therefore, the role of ErbB4 in STAT5A activation may not be STAT5A phosphorylation Tyr-694, but to act as a scaffold for JAK2 and STAT5A, because ErbB4 interacts with each of these proteins.

Additionally, the intracellular domain of ErbB4, with its ability to shuttle between the nucleus and cytoplasm, may act as a nuclear chaperone for STAT5A (32, 41, 42). Because kinase activity of ErbB4 is required for its nuclear localization (32, 45), loss of ErbB4 kinase activity would impair STAT5A signaling in two ways. First, JAK2 induction by ErbB4 ligands (but not in response to PRL) would be compromised in the absence of ErbB4 signaling. Second, loss of ErbB4 kinase activity would inhibit nuclear localization of the ErbB4 intracellular domain and therefore inhibit STAT5A nuclear localization in response to PRL and ErbB4 ligands. Thus, results presented here may explain why loss of ErbB4 signaling in the mouse mammary gland results in impaired differentiation. Previous reports describe the interaction between ErbB4 and STAT5A at the endogenous β-casein promoter in human breast cancer-derived cells. Although STAT5A was detected at the mouse β-casein promoter in HC11 cells by ChIP assay in response to PRL, we were unable to detect ErbB4 at the β-casein promoter in this assay. These results could be a technical issue with the antibodies or conditions used in this analysis. Alternatively, this may suggest that ErbB4 is not a member of the transcriptional complex at the β-casein promoter in HC11 cells.

Although signal transduction in response to PRL and ErbB4 have been studied, relatively little is known about how their respective signaling pathways engage to exert a complex biological outcome like differentiation of the mammary epithelium. Recent studies suggest that PRL and GH may participate in cross talk with EGFR and ErbB2 (22, 23, 24, 51, 52, 53, 54, 55, 56). GH causes EGFR phosphorylation on both tyrosines and threonines in a context-specific manner, resulting in enhanced ERK activation and decreased EGFR degradation as well as enhanced or decreased EGFR signaling, depending on the cellular context (21, 22, 23, 24, 52, 54). Other studies have shown that costimulation of several human breast cancer cell lines with PRL and EGF result in a synergistic increase in cell motility (55).

We show herein that simultaneous activation of PRLR and ErbB4 enhances STAT5A transcriptional activity, a hallmark of lactogenic differentiation. ErbB4 intracellular domain release may be needed for optimal action. Cross talk between PRLR and ErbB family members places JAK2 at a critical position in cell fate decision toward differentiation. Given the opposing roles of ErbB1 (i.e. growth) and ErbB4 (i.e. differentiation) in the mammary epithelium, stimulation of ErbB4 via ErbB4 ligands or by PRL may switch the balance toward differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
HC11 cells and all derivatives were cultured at 5% CO2 in growth medium [DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, EGF (10 ng/ml; Invitrogen, Carlsbad, CA), insulin (5 µg/ml; Sigma-Aldrich, St. Louis, MO), and hydrocortisone (1 µg/ml; Sigma-Aldrich)] unless otherwise indicated. For some experiments, cells were serum starved for 16 h [serum-free/EGF-free DMEM-F12 medium supplemented with hydrocortisone (1 µg/ml) and insulin (5 µg/ml)] and then treated for the indicated time points with ovine PRL (Sigma-Aldrich) or HB-EGF (Sigma-Aldrich), the Erk inhibitor U0126 (10 µM; Promega Corp., Madison, WI), the Src inhibitor PP2 (10 µM; Calbiochem, La Jolla, CA), or the pan-ErbB inhibitor GW572016/lapatinib (GlaxoSmithKline, Research Triangle Park, NC). COS7 cells were purchased from American Type Tissue Culture Collection (Manassas, VA) and were cultured in DMEM supplemented with 10% fetal bovine serum at 5% CO2, unless otherwise indicated.

Western Analysis and Immunoprecipitation
Cells were lysed, and proteins were precipitated with the following antibodies as described previously (57): rabbit polyclonal anti-ErbB1 and -ErbB4 (C terminus) generated in this laboratory and previously described (57); JAK2, PRLR, phosphotyrosine (PY20), Src, and P-Src (Santa Cruz Biotechnologies, Santa Cruz, CA), STAT5A and P-Tyr 694 STAT5 (Zymed Laboratories, San Francisco, CA), p44/42, and phospho-p44/42 (Cell Signaling Technologies, Beverly, MA). For coimmunoprecipitation experiments, cells were cross-linked before lysis using 3,3'-dithiobis[sulfosuccinimidylpropionate] (DTSSP; Thermo Fisher Scientific/Pierce Protein Research Products, Rockford, IL) according to the manufacturer’s directions. Immunoprecipitation samples were analyzed by Western as previously described (57):

Plasmids and Mutagenesis
pcDNA4-HER4 and pcDNA4-HER4KD were constructed by cloning an EcoRI-XbaI fragment of the human ErbB4/HER4 cDNA from pLXSN-HER4 and pLXSN-HER4KD (57) into the multicloning site of pcDNA4/TO (Invitrogen). pRK-STAT5A, pRK5-JAK2WT, and pRK5-JAK2KD were provided by Dr. James Ihle (St. Jude’s Research Foundation, Memphis, TN). JAK2{alpha}s and JAK2vs were provided by Hallger Rui, Georgetown University, Washington, DC (17). pβCasein-lux has been previously described (32). siRNA sequences against mouse JAK2 and mouse ErbB4 were purchased from Dharmacon (Lafayette, CO) and transfected according to the manufacturer’s protocol.

Transfections and Luciferase Assays
A total of 5 x 105 HC11 cells or COS7 cells were seeded into a 10-cm dish 24 h before transfection. Cells were transfected in 5 ml serum-free DMEM with 12 µl Fugene6 transfection reagent (Roche, Indianapolis, IN) and 5 µg plasmid DNA. For generation of stable cell lines, cells were selected with Zeocin (Invitrogen; 1 µg/ml). pRK5-JAK2WT and pRK5-JAK2KD were cotransfected with empty pRK5 and then selected with G418. All analyses were performed on pooled clones (>10 clones). For transient transfection assays, HC11 cells were treated with factors in serum-free medium 4 h after transfection; COS7 cells were treated with factors in 2% serum. Cells were harvested 48 h after transfection. Luciferase assays were performed using 200 µg protein (luciferase assay kit; Promega, Madison, WI) according to the manufacturer’s instructions. To suppress expression of endogenous ErbB4, cells were transiently transfected with ErbB4 siRNA SMARTpool or nonspecific siRNA control pool sequences using siIMPORTER transfection reagent (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s instructions.

ChIP Assays
HC11 cells (107) were cross-linked with 1% formaldehyde in growth medium for 20 min at room temperature on a shaking platform. Cells were washed with ice-cold PBS (pH 7.4) supplemented with phenylmethylsulfonyl fluoride (1 mM), aprotinin (1 µg/ml), and leupeptin (1 µg/ml) (each from Sigma-Aldrich). Cells were lysed in buffer A [1 mM dithiothreitol, 1.5 mM Mg2CL2, 10 mM KCl, 0.1% Nonidet P-40, 25 mM HEPES (pH 7.8)] for 20 min at 4 C, passed through a 30-gauge needle, and centrifuged for 10 min at 3000 rpm at 4 C. Cell pellets were resuspended in buffer B [125 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM HEPES (pH 7.9)]. The suspension was sonicated 10 times in 10-sec bursts followed by 1 min of cooling on ice and then centrifuged at 12,000 rpm for 30 min at 4 C. The supernatants were diluted 10-fold in buffer B supplemented with protease inhibitors. Twenty microliters of the diluted supernatant were stored at –20 C for use as input controls in subsequent PCR after reversal of cross-linking. Anti-STAT5A antibody (1:200; Zymed Invitrogen) and protein A/G+ agarose (Santa Cruz Biotechnologies) were added to the remaining diluted supernatants and then tumbled at 4 C for 3 h. Beads were collected by centrifugation for 20 min at 4 C and washed in buffer B five times at 4 C. Formaldehyde cross-linking were reversed by incubation in buffer B at 65 C overnight, followed by supplementation with proteinase K (5 µg/ml) for the final hour. DNA was recovered by phenol-chloroform extraction and ethanol precipitation, then resuspended in water (10 µl). Each sample was subsequently analyzed by 35 cycles of PCR using the following primers derived from the mouse β-casein gene 5' flanking region, taken from the NCBI genomic database, accession number NT_109320: forward, 5'-GGT GTT TTG TTT CCT ATT-3'; reverse, 5'-TTC AAA AGA GTA CAA CCT-3'. PCR were analyzed on ethidium bromide-stained 2% agarose gels.

Reverse Transcription and Quantitative Real-Time PCR
Total RNA was harvested from HC11 cells with Trizol Reagent (GIBCO Life Science). Total RNA (10 ng) was reverse transcribed with transcript-specific primers and then amplified with transcript-specific primers in the presence of a TAM-labeled, target-specific probe, using the AmpliTaq EZ RT-PCR kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions, with a melting temperature of 62 C. The following primers and probes were used: SOCS1, forward 5'-CCG TGG GTC GCG AGA AC-3', reverse 5'-AAG GAA CTC AGG TAG TCA CGG AGT A-3', and probe 5'-TGG CGC GCA TCC CTC TTA ACC C-3'; SOCS3, forward 5'-CCA CCC TCC AGC ATC TTT GT-3', reverse 5'-CAG GCA GCT GGG TCA CTT TC-3', and probe 5'-ACT GTC AAC GGC CAC CTG GAC TCC T-3'. The cycle threshold (CT) for each transcript within each sample was corrected for the CT of GAPDH within each sample, then normalized to the CT of a single sample, and converted into a relative level of expression using the {Delta}{Delta}CT method, such that all values are presented as a fold change in reference to a single sample. Samples were analyzed six times.


   ACKNOWLEDGMENTS
 
We thank Tona Gilmer (GlaxoSmithKline) for collaboration and provision of GW572016 for these studies.


   FOOTNOTES
 
This work was supported by the Breast Cancer Research Foundation and grants from the National Institutes of Health, GM00678 and CA112553.

Disclosure Statement: This work does not represent any conflicts of interest for any of the authors.

First Published Online July 24, 2008

Abbreviations: ChIP, Chromatin immunoprecipitation; CT, cycle threshold; DTSSP, 3,3'-dithiobis[sulfosuccinimidylpropionate]; EGF, epidermal growth factor; EGFR, EGF receptor; HB-EGF, heparin-binding-EGF; JAK2, Janus kinase 2; PRL, prolactin; PRLR, PRL receptor; SH2, Src homology 2; siRNA, small interfering RNA; SOCS1, suppressor of cytokine signaling 1.

Received for publication February 11, 2008. Accepted for publication July 17, 2008.


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 ABSTRACT
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 DISCUSSION
 MATERIALS AND METHODS
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